Article pubs.acs.org/est
Enhanced Stability and Chemical Resistance of a New Nanoscale Biocatalyst for Accelerating CO2 Absorption into a Carbonate Solution Shihan Zhang, Hong Lu, and Yongqi Lu* Illinois State Geological Survey, Prairie Research Institute, University of Illinois at Urbana−Champaign, Champaign, Illinois 61820, United States S Supporting Information *
ABSTRACT: A novel potassium-carbonate-based absorption process is currently being developed to reduce the energy consumption when capturing CO2 from coal combustion flue gas. The process employs the enzyme carbonic anhydrase (CA) as a catalyst to accelerate the rate of CO2 absorption. This study focused on the immobilization of a new variant of the CA enzyme onto a new group of nonporous nanoparticles to improve the enzyme’s thermal stability and its chemical resistance to major impurities from the flue gas. The CA enzyme was manufactured at the pilot scale by a leading enzyme company. As carrier materials, two different batches of SiO2−ZrO2 composite nanoparticles and one batch of silica nanoparticle were synthesized using a flame spray pyrolysis method. Classic Danckwerts absorption theory with reaction was applied to determine the kinetics of the immobilized enzymes for CO2 absorption. The immobilized enzymes retained 56−88% of their original activity in a K2CO3/KHCO3 solution over a 60-day test period at 50 °C, compared with a 30% activity retention for their free CA enzyme counterpart. The immobilized CA enzymes also revealed improved chemical stability. The inactivation kinetics of the free and immobilized CA enzymes in the K2CO3/ KHCO3 solution were experimentally quantified.
1. INTRODUCTION Anthropogenic carbon dioxide (CO2) is the primary greenhouse gas. Fossil-fuel-fired power plants are major sources of CO2 emission, contributing approximately 40% of total CO2 emissions in the United States. Thus, CO2 capture from these power plants is essential to reduce the CO2 emissions and mitigate the potential for global warming. Absorption-based processes are the most mature option for postcombustion capture of CO2 from coal combustion flue gas. Currently, monoethanolamine (MEA) absorption processes are considered state of the art, but they are very expensive, typically costing from US$50 to 70 per tonne of CO2 avoided.1 The major cost contributor, amounting to approximately 60%, is the parasitic power and steam consumption of the capture process that results in a significant derating (about 30%) of the power plant.2,3 Reducing energy consumption is key to lowering the CO2 capture cost for absorption-based processes, and minimizing the reboiler heat duty or lowering the quality of the extracted steam is the most viable way to reduce the energy used in the process. Recently, a novel integrated vacuum carbonate absorption process (IVCAP) has been proposed to reduce the quality and quantity of heat required for postcombustion CO2 capture.4−6 In the IVCAP, CO2 is absorbed into a potassium carbonate (PC, K2CO3) aqueous solution at a flue gas temperature typically ranging between 40 and 60 °C. Because of the low © 2013 American Chemical Society
alkalinity of K2CO3, the heat of absorption of the CO2/K2CO3 system (609 kJ/kg) is much less than that of the CO2/MEA system (1919 kJ/kg). The weak affinity of CO2 for the K2CO3 solvent allows for CO2-stripping at a relatively low temperature (50−70 °C) and under vacuum pressure conditions (2−8 psia). This indicates that either low-quality steam or waste steam from a power plant, depending on the stripping temperature, can be used as a heat source in the CO2 stripping operation of the IVCAP. Meanwhile, a portion of the steam, due to a low stripping pressure, can be introduced into the stripper for direct heating. This is very different from MEA-based processes where all steam is introduced to the reboiler for indirect heating. As a result, both the quality and quantity of steam required for the CO2 stripping process can be reduced, which will lower the parasitic power losses for a power plant equipped with the IVCAP system compared with the MEA system. However, low alkalinity of a PC system corresponds to a much slower rate of CO2 absorption compared with the MEA-based systems. To address this problem, the enzyme carbonic anhydrase (CA) is introduced to the IVCAP as a biocatalyst to accelerate the Received: Revised: Accepted: Published: 13882
July 19, 2013 November 3, 2013 November 4, 2013 November 4, 2013 dx.doi.org/10.1021/es4031744 | Environ. Sci. Technol. 2013, 47, 13882−13888
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copy (ICP-MS), and a Micromeritics surface-area analyzer. Classic Danckwerts absorption theory with reaction was applied to estimate the kinetic parameters of the immobilized enzymes. The activities of the immobilized ACA1 enzymes were tested and compared with those of their free counterpart. The thermal stability of the immobilized enzymes and their chemical resistance to major flue gas impurities were investigated over a 60-day period at 50 °C and pH 10.5. A kinetic model of enzyme inactivation was applied to describe the activity loss rate of the immobilized ACA1 enzymes under typical IVCAP conditions.
absorption rate while keeping the heat of absorption and phase equilibrium behavior of the system unchanged. The CA enzyme is a zinc metalloenzyme that can efficiently catalyze the hydration of CO2 to form bicarbonate.7 A related technical issue of the IVCAP to be considered is the thermal and chemical stability of the CA enzyme biocatalyst over time because they directly affect the operating cost. In the IVCAP, the CO2 absorption operates at the temperatures ranging from 40 to 60 °C and pHs ranging from approximately 9 to 11. To use the CA enzyme for CO2 capture, its activity and stability performance need to conform to the operation conditions of the IVCAP. Approaches to addressing these concerns include sourcing CAs from thermophilic organisms,8,9 using protein engineering techniques to create thermotolerant enzymes,10 designing CA mimics,11 and immobilizing enzymes.12−14 Immobilization of enzymes on solid carriers is perhaps the most commonly used strategy for improving the operational stability of biocatalysts and increasing the flexibility of reactor design.15,16 The thermal stability of the immobilized enzyme generally is improved by the molecular rigidity introduced by its attachment to a rigid support and the creation of a protected microenvironment. The CA enzyme can be immobilized on either porous materials or nonporous nanoparticles. If necessary, the immobilized enzyme can be separated from the solvent prior to entering a stripper. Our previous studies indicated that the CA enzymes immobilized on both types of materials exhibited improved thermal and chemical stability.17,18 However, the CA enzyme immobilized on porous materials showed a relatively low activity because porous materials incur significant intraparticle diffusion resistance to the CO2 substrate. In contrast, the CA enzyme immobilized onto nonporous nanoparticles achieved a higher retention of enzyme activity because intraparticle diffusion resistance to CO2 substrate in the nonporous nanoparticles is minimal.19 In our previous study, silica nanoparticles were employed as a nonporous support material for enzyme immobilization because of their excellent biocompatibility and hydrophobicity.18 However, silica nanoparticles may gradually dissolve in an alkaline solution,20 such as the K2CO3 solution employed in the IVCAP. As a result, the enzyme may leach from the support material, thus impairing the stability of the immobilized enzyme. Budd and Frackiewicz21 suggested an electrophilic/nucleophilic reaction mechanism for the attack of an alkali on silica-based materials. The attack by OH− results in the destruction of Si−O−Si bonds on the silica surface. Resistance of Si−O−Si to OH− is thus critical to the chemical durability of silica nanoparticles. Because hydration of ZrO2 is more thermodynamically favored than that of SiO2, a thin layer of hydrated ZrO2 can be formed to prevent the OH− from attacking the silica network. The hydrated ZrO2 layer is not soluble in the pH range of 0−17.22 In this study, two batches of SiO2−ZrO2 composite nanoparticles were synthesized by a liquid-fed flame spray pyrolysis (FSP) technique and used as the enzyme immobilization support materials. Pure silica nanoparticles made by the FSP described in our previous work18 also were used as a support material for the purpose of comparison. A new CA enzyme (hereafter termed ACA1) supplied by a leading enzyme company from a pilot-scale production was employed for the immobilization. The physical properties of the silica-based nanoparticles, including their crystalline structure, composition, and surface area, were characterized, respectively, by X-ray diffraction (XRD), inductively coupled plasma mass spectros-
2. MATERIALS AND METHODS 2.1. Materials. The technical-grade ACA1 enzyme was a proprietary enzyme of microbial origin provided by Novozymes A/S, Denmark. The enzyme was produced by fermentation in a benign microbial host and supplied in the form of a partially purified brown aqueous solution containing the dissolved enzyme. The following chemicals were purchased from SigmaAldrich Corporation: γ-aminopropyl triethoxysilane (≥98%), glutaraldehyde solution (25%, w/w), tetraethylorthosilicate (TEOS, 99%), zirconyl 2-ethylhexanoate (EH) in mineral spirits (∼6 wt % Zr), xylene (99%), H4BNa (≥98%), and KBr (FTIR grade, ≥99%). The reagents HNO3, NaH2PO4, Na2HPO4, KHCO3, K2CO3, tris-hydroxymethyl aminomethane, toluene, and acetone were obtained from Fisher Scientific Inc. 2.2. FSP Synthesis of Nanoparticles. All the nanoparticles were synthesized by the FSP method. A detailed description of the FSP experimental setup was provided in our previous work.18 TEOS and xylene were chosen as the precursor and solvent, respectively, for the synthesis of silica nanoparticles (SN1). SiO2−ZrO2 composite nanoparticles were synthesized using a one-step FSP method. Zirconyl EH in mineral spirits and TEOS were used as precursors for zirconia and silica, respectively. The two precursors were dissolved in xylene until the total concentration reached 0.5 M; the concentration of the zirconia precursor varied from 0.1 to 0.25 M. Two batches of SiO2−ZrO2 composite nanoparticles, with Zr/Si molar ratios of 1:4 (SZ1) and 1:1 (SZ2), were synthesized. The other synthesis conditions are shown in Supporting Information (SI) Table SI-1. 2.3. Characterization of Nanoparticles. The Brunauer− Emmett−Teller (BET) surface area of the nanoparticles was determined by N2 adsorption at −196 °C (with a Micromeritics ASAP 2020 analyzer) as described previously.18 Crystalline phases of the nanoparticles were characterized by XRD (with a Siemens-Bruker D5000 X-ray diffractometer).18 Chemical composition analysis of the FSP-made nanoparticles was carried out by fully digesting the particles in aqua regia and hydrofluoric acid. The Si and Zr elemental concentrations of the aliquot were determined with an inductively coupled plasma mass spectrometer (ICP-MS, Thermo Elemental PQ ExCell). In addition, to measure the rate of dissolution of nanoparticles, the concentration of the elemental Si in a 0.1 M KHCO3−K2CO3 solution (pH 10.5) mixed with SN1, SZ1, or SZ2 nanoparticles kept at 50 °C for the specified period was analyzed by ICP-MS. 2.4. Enzyme Immobilization. The ACA1 enzyme was purified by the ammonia sulfate precipitation method before immobilization. A detailed description of the purification method can be found in our previous work.17 13883
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and Cb are the physical solubility of CO2 at the pressure prevailing at the interface and the concentration of unreacted molecular CO2 in the liquid phase, respectively, kmol m−3; kcat is the turnover number of the enzyme, s−1; kH2O (s−1), kOH (m3 kmol−1 s−1), and kE (m3 kmol−1 s−1) are the rate constants for the reactions of CO2 with H2O, OH−, and CA enzyme, respectively; [E] is the concentration of CA enzyme; and Km is the Michaelis constant, that is, the CO2 substrate concentration at which the reaction takes place at half the maximum rate achieved by the system, kmol m−3. Note that compared with the other two terms, the value of kH2O (in a range between 0.02 and 0.0375 s−1 at 25 to 50 °C) is negligible.26,27 The concentration of CO2 in the alkaline solution (Cb) is negligible. Thus, by combining eq 4 and eq 2 and integrating, we get
Immobilization of the CA enzyme onto FSP-made SN1, SZ1, and SZ2 nanoparticles followed a covalent coupling-based method developed in our previous studies.17,18 The method consists of activating the surface of the nanoparticles with γaminopropyl triethoxysilane and glutaraldehyde solutions, and then covalent coupling of the CA enzyme with the activated surface.18 It generally involved 10 mL of 500 mg/L ACA1 enzyme coupling with 0.05−0.1 g of the activated nanoparticles in a 0.1 M phosphate buffer (pH 8.05) at room temperature for 1.5 h. Details of the enzyme immobilization procedure are available elsewhere.18 In this study, three immobilized enzyme samples were prepared: ACA1 on SN1 (ACA1−SN1), ACA1 on SZ1 (ACA1−SZ1), and ACA1 on SZ2 (ACA1−SZ2). The products were stored in a 0.1 M phosphate buffer (pH 8.05) at 4 °C before conducting the activity and stability measurements. The amount of the ACA1 enzyme loaded on the nanoparticles was determined by measuring the enzyme concentrations in the solution before and after the immobilization treatment. The measurement of enzyme concentration was referred to the method developed by Bradford. 23 2.5. Enzyme Activity Assay. The activities of the free and immobilized ACA1 enzymes for CO 2 hydration were determined by a modified manometric method in a batch stirred-tank reactor (STR).24 A detailed description of the method can be referred to our previous work.17 A typical test involved the absorption of CO2 with a partial pressure of 0.76 kPa into 15 mL of a 0.1 M KHCO3−K2CO3 solution (pH 10.5) with a desired dosage level of the enzymes at 4 °C and under a vacuum condition. The CO2 pressure during the test was determined by a vacuum pressure transducer (Omegadyne, PX429-012AUSB) and was used to calculate the instantaneous rate of CO2 absorption. It should be noted that control tests were also conducted following the above experimental procedure. The nanoparticles alone revealed no catalytic activity for CO2 hydration. The kinetic parameters of the free and immobilized ACA1 enzymes were calculated according to classic Danckwerts absorption theory with reaction.18 For CO2 absorption undergoing a pseudo-first-order reaction and taking place in the STR, the following equation could be derived based on the mass conservation principle, ideal gas law, and Henry’s law:25 R=−
⎛ Pi ,0 ⎞ ln⎜ ⎟ = ⎝ Pi ⎠
kE =
and
kov = k H2O + k OH[OH−] + kE[E]
kE =
kcat Km
(5)
2 mCA − m02 D[E]
(6)
where mCA and m0 are the slopes of the lines obtained by the plotting of Ln((Pi,0)/(Pi)) versus (RTA)/(VGHe)Δt for the absorption of CO2 into PC solutions with and without the CA enzyme, respectively. The value of kE can thus be determined when D and [E] are known. The dilute buffer solution (0.1 M KHCO3−K2CO3) used in this assay has a viscosity similar to that of pure water. Thus, the diffusivity of CO2 in the buffer solution is assumed to be the same as that in water. The correlations given by Versteeg and Van Swaalj28 were used to calculate the diffusivity and solubility of CO2 in pure water. The method developed by Weisenberger and Schumpe29 was used to estimate He for CO2 in the KHCO3−K2CO3 solution. An immobilization factor (IF) was used in this study to compare the enzymatic activity among different immobilized enzymes. The IF is defined as the ratio of the activity (indicated by kcat/Km) of an immobilized CA with respect to that of its free homogeneous counterpart. The value of the IF is thus also an indication of the retention rate of CA enzyme activity during immobilization. 2.6. Thermal and Chemical Stability Assay. The immobilized ACA1 enzymes (ACA1−SN1, ACA1−SZ1, and ACA1−SZ2) were stored in a 0.1 M KHCO3−K2CO3 buffer solution (pH 10.5) at 50 °C to assess their thermal stability. The activities of the immobilized enzymes were measured periodically at 4 °C over a period of 60 days. The effects of sulfate, nitrate, and chloride ions, three major impurities typically found in blowdown of a flue gas desulfurization scrubber, on the stabilities of the immobilized enzymes also were investigated. During this test, the immobilized ACA1 enzymes were stored at 50 °C in a 0.1 M KHCO3−K2CO3 buffer solution (pH 10.5) with additions of 0.4 M of SO42−, 0.05 M of NO3−, and 0.3 M of Cl−. The activities of the immobilized enzymes were measured periodically at 4 °C over a period of 60 days. The thermal and chemical stabilities of the free ACA1 enzymes also were investigated under the same conditions and served as the baseline for the comparison.
⎛ VG dPi Dk ⎞ = kL ⎜1 + 2ov ⎟ × (C* − C b) A × R gasT dt kL ⎠ ⎝
Pi He
RTA Δt VGHe
where Pi,0 is the initial partial pressure of CO2 in the gas phase, Pa. By rearranging eq 3 and eq 5, we get
(1)
C* =
kL2 + Dkov
(2) (3)
(4)
where R is the rate of CO2 absorption per unit of interfacial area, kmol m−2·s; VG is the volume of the gas phase, m3; A is the gas−liquid interfacial area, m2; Rgas is the universal gas constant, Pa m3 kmol−1 K−1; T is the temperature, K; Pi is the partial pressure of CO2, Pa; t is the time, s; kov is the overall first-order rate constant, s−1; kL is the physical mass transfer coefficient in the liquid phase, m s−1; D is the diffusivity of CO2, m2 s−1; He is Henry’s law constant for CO2, Pa m3 kmol−1; C* 13884
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2.7. Enzyme Inactivation Kinetics. Enzyme inactivation can be expressed schematically as follows:30 ku
for the pure silica nanoparticles was more prominent, indicating a substantial SiO2 content. The sharp peaks of the two SiO2− ZrO2 samples indicate that zirconia was present as a tetragonal crystalline phase (pdf card 80-784). Elemental analysis of the SiO2−ZrO2 nanoparticles by ICP-MS confirmed that the Zr/Si ratios in the FSP nanoparticles were almost identical to those of their liquid precursors (SI Table SI-1). The N2 adsorption−desorption isotherms of the FSP-made nanoparticles were of type IV, as shown in Figure 2. These
ki
N⇔U→I kf
(7)
where N is the native enzyme, U is the unfolded enzyme, and I is the inactive enzyme. ku and kf are the unfolding and refolding kinetic constants, d−1, and ki is the irreversible inactivation kinetic constant, d−1. As shown in eq 7, enzyme inactivation can be described by a reversible unfolding reaction followed by an irreversible reaction. The latter reaction leads to complete inactivation of the enzyme. Generally, enzyme inactivation follows first-order kinetics. The rate of inactivation is determined by the rates of the unfolding and inactivation reactions:31 −
d(N + U ) = kobs(N + U ) dt
(8)
k
kobs =
k i ku f
1+
ku kf
at [N + U ]t = = exp( −kobst ) a0 [N + U ]0
(9)
(10)
where at is the residual enzyme activity, a0 is the initial activity (expressed as kcat/Km), kobs is the apparent inactivation rate constant, d−1, and can be obtained according to eq 10. The above model was applied to describe the activity losses of the immobilized ACA1 enzymes during the tests of thermal and chemical stability.
Figure 2. N2 adsorption isotherms (−196 °C) of the tested FSP nanoparticles.
isotherms indicate a multilayer formation after completion of the monolayer at a relative pressure of approximately 0.2 for the silica nanoparticles and 0.07 for SiO2−ZrO2 composite nanoparticles. The results revealed that all of the FSP nanoparticles were nonporous and that N2 adsorption occurred on the external surfaces of the particles and in the voids between the particles. The isotherms also exhibited a weak adsorption−desorption hysteresis arising from the voids between the nanoparticles. The BET surface area of SN1 was 184 m2/g. The BET surface areas of SZ1 and SZ2 were almost the same (∼120 m2/g). The diameters of the SN1, SZ1, and SZ2, based on their BET surface areas and assuming spherical particles and a density difference, were estimated as approximately 15, 20, and 15 nm, respectively. The concentration of elemental Si in the carbonate/ bicarbonate solution at 50 °C and pH 10.5 resulting from the dissolution of silica from the FSP nanoparticles was determined periodically by ICP-MS, as shown in SI Figure SI-1. For the SN1, SZ1, or SZ2 nanoparticles, most of the silica dissolution occurred in the first 11 days. After that point, almost no more silica was dissolved over the duration of the 60-day test (SI Figure SI-1). The higher the content of zirconia present in the nanoparticles, the less silica was dissolved, indicating that the doping of zirconia mitigated the dissolution of the silica-based nanoparticles into the alkaline solution. 3.2. Enzyme Immobilization onto the SiO2−ZrO2 Composite Nanoparticles. Results of the enzyme loading levels and activities for the immobilized ACA1 enzymes are shown in Table 1. Silica-based nanoparticles possess a large number of hydroxyl groups on the surface and are highly biocompatible with enzymes. The enzyme loading levels on the SN1, SZ1, and SZ2 supports reached 50.2, 35.9, and 22.7 mg/g, respectively. In general, for nonporous nanoparticles with
3. RESULTS AND DISCUSSION 3.1. Characterization of the Nanoparticle Support Materials. The silica nanoparticle (SN1) derived from the TEOS precursor was white, whereas the SiO2−ZrO2 composite nanoparticles (SZ1 and SZ2) were yellow and brown. The XRD patterns of the particles are shown in Figure 1. Incorporation of zirconyl EH and TEOS into the liquid precursor at Zr/Si ratios of 1:1 and 1:4 did not result in the formation of silicates and zirconates despite the high temperature of the flame. An amorphous SiO2 hump (20−25°) was observed for both of the SiO2−ZrO2 particles, but this hump
Figure 1. XRD patterns of the tested FSP nanoparticles. 13885
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that would enhance the rate of protein deactivation. Thus, it is believed that an optimal dosage level of zirconia exists for the activity of the immobilized enzyme on the composite nanoparticles. 3.3. Thermal Stability of the Immobilized Enzymes. Results from testing the thermal stabilities of the immobilized and free ACA1 enzymes in the 0.1 M K2CO3/KHCO3 solution for 60 days at 50 °C are shown in Figure 3. As reported in our
Table 1. Enzyme Loading Levels, Kinetic Parameters, and IF Values of the Immobilized ACA1 Enzymes sample ACA1− SN1 ACA1− SZ1 ACA1− SZ2
enzyme loading, mg/g of support
enzyme loading, mg/g of SiO2
kcat/Km × 10−7, M−1 s−1
IF, %
50.2
50.2
0.297
37.5
35.9
54.4
0.328
41.2
22.7
69.2
0.259
32.5
similar surface properties, the enzyme loading depended on only the specific surface area (completely contributed by the external surface). Because the BET surface area of SN1 was the greatest among the three carriers, they achieved the highest ACA1 enzyme loading. Although the BET surface areas of SZ1 and SZ2 nanoparticles were comparable, the enzyme loading onto SZ1 was higher than that onto SZ2. For the composite nanoparticles, the doped metal oxide phase, rather than being embedded in the silica nanoparticle, would be expected to be segregated to the edge of the nanoparticle at a high metal/Si molar ratio.32 Compared with the SZ1 nanoparticles, the SZ2 nanoparticles were derived from the precursor with a higher Zr/Si molar ratio (1:1). As a result, more of the external surface of the SZ2 nanoparticles was occupied by ZrO2, which was inactive for enzyme immobilization based on the current immobilization method. Thus, the enzyme loading onto the SZ2 particles was much lower than that onto SZ1 despite the comparable surface areas of the two support materials. For the free ACA1, the enzyme activity (described as the value of kcat/Km) was 0.795 × 107 M−1 s−1 at 4 °C. As shown in Table 1, the ACA1−SZ1 exhibited a slightly higher activity retention compared with the ACA1−SN1. Losses of enzyme activity after binding to the nanoparticles are usually attributed to unfavorable protein−protein interactions, protein−nanoparticle interactions, or both. The isoelectric points of silica and the CA enzyme are approximately pHs 2 and 6, respectively. Under the current enzyme immobilization conditions (pH 8.05), both silica and the ACA1 enzyme incurred a negative charge on the surface. In addition to unfavorable protein− protein interactions in the ACA1−SN1, such unfavorable protein−nanoparticle electrostatic repulsions would result in transformation of the native-like structure of the enzyme, thus impairing enzyme activity. With the doping of zirconia into the silica nanoparticles, the negative surface charge of the SZ1 nanoparticles would be lower than that of SN1. Therefore, the electrostatic repulsions between the enzyme and SZ1 nanoparticles would be reduced, resulting in a higher enzyme activity for the ACA1−SZ1 than for the ACA1−SN1. However, increasing the zirconia content in the composite nanoparticles (SZ2) did not result in an increase in enzyme activity retention. By contrast, the ACA1−SZ2 exhibited the lowest enzyme activity among the three immobilized enzymes (Table 1). As discussed, a portion of the surface of the composite nanoparticles was occupied by ZrO2, resulting in less SiO2 on the surface. For the ACA−SZ2, the enzyme loading per unit mass of SiO2 was estimated to be 69.2 mg/g of SiO2 according to the data in Table 1, compared with 50.2 mg/g of SiO2 for the ACA1−SN1 and 54.4 mg/g of SiO2 for the ACA1−SZ1. This suggests that the ACA1−SZ2 had the greatest enzyme coverage on the surface occupied by SiO2. At a higher surface coverage, the enzyme molecules are closer together, thereby leading to a greater likelihood of unfavorable protein−protein interactions
Figure 3. Thermal stability of immobilized ACA1 enzymes in a KHCO3/K2CO3 solution at 50 °C for 60 days.
previous work,18 the free ACA1 enzyme revealed better thermal stability than the standard commercial CA enzyme from SigmaAldrich (SCA). As shown in Figure 3, the free ACA1 retained approximately 60% of its original activity after 30 days at 50 °C and retained approximately 30% of its activity after 60 days. In comparison, the free SCA enzyme lost almost all its original activity after only 30 days at 50 °C.18 The thermal stability of the ACA1 enzyme was substantially improved via immobilization. The ACA1 enzymes immobilized on the nanoparticle support materials (ACA1−SZ1, ACA1−SZ2, and ACA1−SN1) retained 88, 70, and 56%, respectively, of their original activities after 60 days at 50 °C. In particular, the ACA1−SZ1 showed no decrease in activity during the first 30 days at 50 °C. The experimental results were also fit to the enzyme inactivation model described in eq 10. It should be noted that the activity of the immobilized enzymes increased by approximately 20% compared with the initial activity after being stored in the carbonate solution at 50 °C for 1 week (Figure 3). Thus, the activity of the immobilized enzymes after storage for 1 week was adopted as the initial activity to determine the inactivation kinetics of the enzymes. Results showed that the apparent inactivation rate constants (kobs) of the ACA1−SZ1, ACA1−SZ2, and ACA1−SN1 decreased by 61, 44, and 29%, respectively, compared with that of the free ACA1 enzyme (Table 2). Furthermore, the half-life times (t1/2, the time taken for the enzyme activity to reduce to one-half of its initial activity) of the ACA1−SZ1, ACA1−SZ2, and ACA1−SN1 increased by 1.6, 0.8, and 0.4 times compared with the free ACA1 enzyme. It is clear that the thermal stability of the ACA1 enzyme was markedly improved by being immobilized on the nanoparticles. The results also indicate that the ACA1 enzyme immobilized on the SiO2−ZrO2 composite nanoparticles (ACA1−SZ1 and ACA1−SZ2) was more stable than the enzyme immobilized on the pure silica nanoparticle (ACA1−SN1). Two factors might 13886
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Table 2. Inactivation Rates of the Free and Immobilized ACA1 Enzymes without impurities
with impurities
sample
kobs, d−1
R2 value
kobs, d−1
R2 value
free ACA1 ACA1−SN1 ACA1−SZ1 ACA1−SZ2
0.0194 0.0138 0.0075 0.0108
0.97 0.96 0.91 0.95
0.0221 0.0183 0.0102 0.0133
0.95 0.98 0.99 0.99
contribute to improvements in the thermal stability of the enzyme: (1) the addition of Zr(IV) might have reduced the enzyme-support electrostatic repulsions and thus increased the stability of the immobilized enzyme, and (2) the doping of ZrO2 somehow prevented the dissolution of SiO2 in the alkaline solution, which reduced the loss of enzyme from the nanoparticles over time. The results further indicate that an optimal content of ZrO2 in the composite material may exist, based on the fact that the ACA1 enzyme immobilized on the SZ2 support (ACA1−SZ2), which contained more ZrO2 than the SZ1 support, showed a lower thermal stability than the ACA1 enzyme immobilized on the SZ1 support (ACA1−SZ1). 3.4. Chemical Stability of the Immobilized Enzymes. The formation of the coordinated OH− ion on the zinc active site of the CA enzyme plays a key role in catalyzing CO2 hydration. Most monovalent anions are regarded as an inhibitor for CA enzyme because they can bind to the zinc active site of the CA enzyme and thus prevent the formation of OH− coordination.33 In comparison, most divalent anions, such as SO42−, do not inhibit CA activity or they act only as very weak inhibitors.34 However, potential inhibition effects of these anions depend on many factors, such as the type of CA enzyme and the solution conditions applied. The SO42−, NO3−, and Cl− anions were the major impurities in the CO2 solvent as derived from SO2, NOx, and HCl in the coal combustion flue gas. For example, in a typical blowdown from a wet flue gas desulfurization unit, the concentration of SO42− ranges from 0.03 to 0.05 M, NO3− ranges from 0.005 to 0.02 M, and Cl− ranges from 0.3 to 0.7 M.35 The selected concentration levels of the major impurities (0.4 M SO42−, 0.05 M NO3−, and 0.3 M Cl−) thus were comparable to, or exceeded, the those typically derived from the flue gas. As shown in Figure 4, the free ACA1 enzyme retained approximately 28% of its initial activity for CO2 absorption after 60 days at 50 °C, whereas the ACA1−SZ1, ACA1−SZ2, and ACA1−SN1 enzymes retained, respectively, 66, 54, and 41% of their initial activities under the same conditions. As shown in Table 2, in the presence of the anion impurities, the apparent inactivation rate constant (kobs) of the free ACA1 was determined to be 0.0221, and those of the ACA1−SZ1, ACA1−SZ2, and ACA1−SN1 were determined to be 0.0102, 0.0133, and 0.0183 d−1, respectively. In comparison, the kobs values of ACA1−SZ1, ACA1−SZ2, and ACA1−SN1 in the absence of the anion impurities were estimated to be 0.0075, 0.108, and 0.138 d−1, respectively, indicating that the chemical stability of the immobilized ACA1 enzymes was adversely affected, by 23 to 36% over 60 days, in the presence of the anion impurities. It should be noted that the total ionic strength of the KHCO3−K2CO3 buffer solution in the presence of impurities was five times higher than that in the pure KHCO3− K2CO3 buffer. Thus, besides the inhibition by the anions, the adverse effect of ionic strength on the enzyme activity might not be completely excluded. However, the chemical stability of
Figure 4. Relative activity of free and immobilized ACA1 enzymes in a KHCO3/K2CO3 solution in the presence of SO42−, NO3−, and Cl− at 50 °C for 60 days.
the ACA1 enzyme was still significantly improved via immobilization. The half-life times of ACA1−SZ1, ACA1− SZ2, and ACA1−SN1 increased by 120, 70, and 20%, respectively, compared with that of the free ACA1 enzyme in the presence of the anion impurities. In summary, the loading of the ACA1 enzyme onto the silicabased nanoparticles via immobilization decreased as the zirconia content in the nanoparticles increased. The ACA1 immobilized on the SZ1 support with a Zr/Si ratio of 1:4 exhibited the highest enzyme activity compared with the ACA1 on the SN1, which contained no zirconia, and the ACA1 on the SZ2, which had a Zr/Si ratio of 1:1. An optimal content of ZrO2 in the SiO2−ZrO2 composite material yielding the best enzyme activity appears to exist. The thermal stability of the enzyme was significantly improved by immobilization. Compared with the half-life time of the free enzyme counterpart, the half-life times of the immobilized enzymes were increased by up to 1.6 times over the 60-day test period at 50 °C. The ACA1 enzyme immobilized on the SiO2−ZrO2 composite nanoparticles also exhibited improved chemical resistance to the major flue gas impurities. Because of its high activity and improved stability, the ACA1 enzyme immobilized on SiO2−ZrO2 composite nanoparticles is a promising biocatalyst for application in a carbonate-based CO2 absorption system.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in the text. This information is available free of charge via the Internet at http://pubs.acs.org/.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +1 217 244 4985; fax: +1 217 333 8566; e-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful for funding support for this work from the U.S. Department of Energy/National Energy Technology Laboratory through Cooperative Agreement DE-FC2608NT0005498. The Carbonic Anhydrase enzyme was provided 13887
dx.doi.org/10.1021/es4031744 | Environ. Sci. Technol. 2013, 47, 13882−13888
Environmental Science & Technology
Article
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by Novozymes A/S, Denmark. Characterization of synthesized nanoparticles was performed in part at the Frederick Seitz Materials Research Laboratory Central Facilities, University of Illinois at Urbana−Champaign. We also thank Dr. Jonathan H. Goodwin and Ms. Susan Krusemark for their reviews of the manuscript and input. Publication of this paper was authorized by the Director of the Illinois State Geological Survey at the University of Illinois at Urbana−Champaign.
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